Human Serum Albumin (HSA) is 66.4 kDa abundant protein in human serum (50% of total protein) composing of 585 amino acids with the heart shape structure (Sugio, Protein Eng, Vol. 12, 1999, 439-446). Multifunctional HSA protein is associated with its structure that allowed to bind and transport a number of metabolizes such as fatty acids, metal ions, bilirubin and some drugs (Fanali, Molecular Aspects of Medicine, Vol. 33, 2012, 209-290). HSA concentration in serum is around 3.5-5 g/dL. Abnormal HSA level is resulting in abnormal function in human system and can be an indicator for some diseases. The high HSA level can be found in heart failure condition, Alzheimer and diabetes mellitus (Fanali, Molecular Aspects of Medicine, Vol. 33, 2012, 209-290).
Glycated human serum albumin (GHSA) is glycation product of HSA protein, in which glucose sugar is non-enzymatically added on some amino acids (Lysine 199, 281, 439 etc.) of the HSA molecule (Fanali, Molecular Aspects of Medicine, Vol. 33 2012, 209-290). GHSA can be produced in condition with the high level of sugar concentration, which usually found in diabetes mellitus patients. Adding sugar on the GHSA molecule results in three-dimensional structure changes and interferes normal HSA protein functions, for examples lower binding affinity to bilirubin (up to 50%) and cis-parinaric acid (up to 20 times) (Shaklai, Journal of Biological Chemistry, Vol. 259, No. 6, 1984, 3812-3817). Therefore, GHSA level can be an indicator for diabetes mellitus complications and Alzheimer diseases (Shuvae, Neurobiology of Aging, Vol. 22, No. 3, 2001, 397-402). In addition, GHSA level is correlated with blood sugar and glycated hemoglobin (HbAlc) and its half-life is shorter than HbAlc, therefore GHSA level can be the better indicator for diabetes mellitus detection and monitoring (Wincour, Clinical Biochemistry, Vol. 22, 1989, 457-461, Worner, International Journal of Pharmacology, Therapy, and Toxicology, Vol. 31, No. 5, 1993, 218-222).
In case of diabetic nephropathy, GHSA will interact with receptor in the mesangial cells, which are associated with the glomerular dysfunction (Cohen, Clinical and Methodological Aspects. Diabetes Technology & Therapeutics, 1999, Thomas, Journal of 10 American Society of Nephrology, Vol. 16, 2005, 2976-2984, Ziyadeh, Kidney International, Vol. 53, 1998, 631-638). In 1994 and 1995, Cohen and colleagues found that monoclonal antibody that specifically bound GHSA could retard the progression of diabetes nephropathy in mice and prevent the GHSA from causing further harm in the kidney (Cohen, U.S. Pat. No. 5,518,720). Therefore this antibody have a potential for drug development in diabetes nephropathy complication.
It has been found that GHSA is associated with the protein phosphorylation in retinal cell growth, resulting in diabetes retinopathy (Okumura, Journal of Opthalmology, Vol. 51, 2007, 231-243). In 2007, Higashimoto and his colleagues selected single stranded DNA (ssDNA) that specifically bound to GHSA in vitro and they also found that some selected aptamers could inhibit GHSA toxicity in retinal pericytes (Higashimoto, Microvascular Research, Vol. 74, 2007, 65-69, Inou, US patent number US/2009/0023672 A1), which can be developed for the anti-diabetes retinopathy drugs.
Human Serum Albumin Detection
                1. Dye-Binding Method: There are 2 types of GHSA detection by dye, Which are Bromcresol Green (BCG) and Bromcresol Purple (BCP).                    Bromcresol Green: In 1965, it has been proved that bromcresol green, which is negative charge molecule could bind HSA protein at pH 7-7.1. The absorbance of the binding complex could be detected by spectrophotometry at the absorption wavelength 615 nm. Increasing of HSA concentration is associated with decreasing of 615 nm absorption (Rodkev, Clinical Chemistry, Vol. 11, No 4, 1965). In 1976, BCG has been found to bind to other proteins (α- and β-globulin) in condition with the low HSA for examples kidney failure and dialysis patients (Gustafsson, Clinical Chemistry, Vol. 22, No. 616, 1976, Webster, Clinica Chimica Acta, Vol. 53, No. 109, 1974). Therefore, BCG can be only used for screening method.            Bromcresol Purple: Detection of HSA using BCP method was firstly used in 1970 by Louderback and his colleagues (Louderback, Clinical Chemistry, Vol. 14, 1970, 793-794) and future developed by Carter and his colleagues (Carter, Microchem Journal, Vol. 15, 1970, 531-539). In 1978, Andrew and colleagues invented automate system based on BCP method (Andrew Clin Chem, Vol. 24., No. 1, 1978, 80-86). The principle of BCP method is based on BCP charge, which is the higher positive charge dye than the BCG charge. The BCP can specifically bind to HSA, leading to more broaden absorption wavelength comparing with BCG method. In addition, BCP method can be used for detection of human serum albumin in the lower concentration. Therefore, BCP method is more popular method than BCG method. However, it has been reported that 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), which can be found in kidney failure patients who have been done dialysis for a long period of time, interferes BCP method (Basil Clinical Chemistry, Vol. 55, No. 3, 2009, 583-584). Therefore, it is better to develop new method for HSA measurement that can be used in kidney failure patients and other abnormal conditions.                        2. Immunochemical Assay: Immunochemical assay seems to be the most sensitive and specific method for HSA detection. Principle of the assay is depending on the affinity binding of HSA and antibody. The final products could be measured by detection of the turbidity, fluorescence intensity and UV absorption (Basil, Clinica Chimica, Vol. 258, 1997, 3-20). In order to get an accurate results, the assay requires several sample dilutions, leading to high cost and time consuming. However, this assay is suitable for detection of low HSA concentration in urine and other secretions.Glycated Human Serum Albumin Detection        
Nowadays, glycated human serum albumin detection is based on the binding of boronic acid and cis-diol group of the glucose molecule on the GHSA protein. The most three popular methods are described below.                1. Boronate Affinity Chromatography (BAC): Boronic acids, which are coated on the resin beads, will bind to glucose molecule on GHSA protein in the sample. Then the unbound molecules will be washed out and the remaining GHSA protein will be analyzed by measuring the absorption of tryptophan amino acids.        2. Enzyme Link Boronate Immunoassay (ELIBA): Antibodies against HSA protein will bind to both HSA and GHSA protein. After the binding of Horseradish Peroxidase (HPR) conjugated boronic molecule and cis-diol group on the GHSA protein, GHSA concentration can be analyzed using the similar method as ELISA (Ikeda, Clinical Chemistry, Vol. 44, No. 2, 1998, 256-263).        3. Enzymatic Assay: Amino acids with the glucose attachment will be digested by proteinase enzyme, resulting in single glycated amino acids. Then glycated amino acids will be oxidized by Ketonamine oxidase enzyme, leading to the formation of hydrogen peroxide. The amount of hydrogen peroxide molecule, which correlated with the concentration of GHSA, can be measured using peroxidase method. On the other hand, total HSA can be analyzed using BCP method as previously described and the percentage of glycation can be calculated (Kohzuma, Journal of Diabetes Science and Technology, Vol. 5, No. 6, 1455-1462).        
Previous HSA and GHSA detections are suitable for only screening method because they are lacking of specificity. The ideal method should be more specific, which is depending on the affinity binding of the specific binding molecules (antibody or aptamer) and HSA/GHSA.
Aptamers Against Human Serum Albumin and Glycated Human Serum Albumin
Aptamer is a short ssDNA or RNA that specifically bind to target molecule using three-dimensional structure. Target molecules could be cells, proteins, metal ions, and toxin. The aptamer can be selected from the large aptamer library using the method called “Systematic Evolution of Ligands by Exponential Enrichment” or “SELEX” (Tuerk, Science, Vol. 249, 1990, 505-510, Ellington, Nature, Vol. 346, 1990, 818-822). The principle of the SELEX method is the repeating of aptamers selection against target molecule. The higher pressure condition will be added to each selection process to obtain higher specific binding aptamers. Then the selected aptamers will be amplified and the process will be repeated until the affinity binding of selected aptamer is constant.
Aptamer is similar as antibody, in which they can bind specifically to target molecule. However, aptamer is more stable and easily to produce comparing with the antibody. It has been reported that aptamers could be developed and used as a drug, drug delivery and applied for diagnostic field (Kyung-Mi Song, Sensors, Vol. 12, 2012, 612-631). Aptamers against GHSA have been reported in 2007 by Higashimoto and colleagues. They also found that some selected aptamers could inhibit toxicity of GHSA in retinal pericyte (Higashimoto, Microvascular Research, 2007, 65-69, US patent number US 2009/0023672 A1).
The present invention is about aptamers against HSA and GHSA. Selected aptamers in this invention have higher binding affinity than that from the previous report and have a potential to be used in the diagnostic field and also drug development.